1. Field of the Invention
This invention relates to the inhibition of angiogenesis, particularly the inhibition of angiogenesis in angiogenesis-associated conditions including, without limitation, neoplasia, rheumatoid arthritis, endometriosis, psoriasis, and vascular retinopathies.
2. Summary of the Related Art
The process of angiogenesis results in the formation of new blood vessels. During the process of angiogenesis, endothelial cells which exist in a quiescent state as part of an existing blood vessel grow and enter a migratory, proliferative state. This migratory, proliferative state of endothelial cells undergoing angiogenesis is eventually resolved when the cells return to the quiescent state as part of a functional new blood vessel. The process of angiogenesis is orchestrated by a complex network of multiple macromolecular interactions. Some essential angiogenic factors include fibroblast growth factor-basic (bFGF), vascular endothelial growth factor (VEGF), the Angiopoietins (Ang xe2x88x92xc2xd/3/4), cytokines, extracellular matrix (ECM) proteins, and matrix metalloproteases (MMP). These factors are produced locally by stromal cells (e.g., smooth muscle cells, pericytes, fibroblasts) and by activated leukocytes that are recruited to the area (Risau, W. (1997) Nature 386(6626):671-674; Risau and Flamme (1995) Ann. Rev. Cell Dev. Biol. 11:73-91).
The interplay of growth factors (e.g., VEGF and Ang-2) and surface protein-ECM interactions (e.g., xcex1vxcex23/5 interactions with both collagen and matrix metalloprotease-2) drive the process of angiogenesis through a predictable sequence of events. Activation of endothelial cells by pro-angiogenic stimuli results in vasodilation, hyperpermeability, and local release of proteases which degrade the basement membrane and ECM. This allows the formation of a provisional fibrin matrix, which provides a primary scaffold for the assembly of early microvessels. Motogenic endothelial cells sprout into the matrix and migrate with controlled matrix degradation at the tip. Proliferation occurs proximal to migration with formation of a primitive tube. Extensive remodeling ensues until the new capillary matures and anastomoses (i.e., fuses and joins) with other sprouts (Risau, W. (1997) Nature 386(6626):671-674; Risau and Flamme (1995) Ann. Rev. Cell Dev. Biol. 11:73-91).
Angiogenesis is stimulated and harnessed by some neoplasms (e.g., tumors) to increase nutrient uptake. However, in contrast to normal angiogenesis, which leads to anastomoses and capillary maturation, angiogenesis associated with neoplasia is a continuous process. Endothelial cells are activated by nearby neoplastic cells to secrete not only VEGF which stimulates angiogenesis, but also matrix metalloproteases (MMP) which degrade the surrounding extracellular matrix. The endothelial cells then invade the extracellular matrix where they proliferate, migrate, and organize to form new blood vessels, which support neoplasm growth and survival.
The newly vascularized neoplasm continues to grow, leading to further nutrient deprivation and chronic pro-angiogenic signaling. The vasculature of neoplasms is characterized by the presence of lacunae and a low rate of anastomosis. This partially dysfunctional vasculature fuels the permanent requirement for angiogenesis. Additionally, this incomplete vasculature allows the shedding of neoplastic cells into the systemic circulation. Hence, the angiogenic potential of a neoplasm correlates with metastatic potential (Weidner et al. (1991) N. Engl. J. Med. 324(1):1-8; Folkman and Shing (1992) J. Biol. Chem. 267(16):10931-10934). As a significant proportion of neoplasms are dependent on continued angiogenesis, inhibition of angiogenesis blocks neoplasm growth which often leads to complete necrosis of the neoplasm. (Weidner et al. (1991) N. Engl. J. Med. 324(1):1-8; Folkman and Shing (1992) J. Biol. Chem. 267(16):10931-10934). Thus, methods or reagents for inhibiting angiogenesis associated with neoplasia could represent a viable anti-neoplasia therapy.
Other disease conditions are characterized by aberrant levels of angiogenesis. For example, rheumatoid arthritis (RA) is an inflammatory disease associated with intense angiogenesis (see, e.g., Jackson et al. (1988) Ann. Rheum. Dis. 57(3):158-161). Another angiogenesis-associated condition is psoriasis, a chronic skin disorder that affects one in fifty people world wide and over five million people in the United States. The most common form of the disease is called plaque psoriasis or psoriasis vulgaris. Other forms are pustular, guttate, inverse, and erythrodermic psoriasis.
Yet another angiogenesis-associated condition is vascular retinopathy, which includes diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity (ROP), exudative age-related macular degeneration, sickle cell retinopathy, and radiation retinopathy (see, e.g., Aiello, L. P. (1997) Curr. Opin. Ophthalmol. 8(3):19-31; Pierce et al. (1994) Int. Ophth. Clinics 34:121-148). An additional angiogenesis-associated condition is endometriosis (see, e.g., Abulafia and Sherer (1999) Obstet. Gynecol. 94(1):148-153; Healy (1998) Hum. Reprod. Update 4(5):736-740). Thus, there exists a need to control angiogenesis as a means for treating and/or alleviating the symptoms of angiogenesis-associated conditions.
There are a number of known reagents that inhibit angiogenesis that have been used for various conditions associated with angiogenesis. For example, O""Reilly et al. teach that isolated endostatin protein is allegedly an inhibitor of endothelial cell proliferation and angiogenesis (O""Reilly et al., U.S. Pat. No. 5,854,205). Another compound with alleged anti-angiogenesis activity is heparin or heparin fragments (Folkman et al., U.S. Pat. No. 4,994,443). O""Reilly et al. also teach that angiostatin can allegedly reversibly inhibit proliferation of endothelial cells (O""Reilly et al., U.S. Pat. No. 5,733,876). However, many of the known anti-angiogenesis reagents and therapies have limitations. For example, for ROP, a common cause of blindness in children, two therapeutic methods, cryotherapy and laser therapy, are not completely effective and themselves cause damage to the eye, resulting in a reduction of vision (Pierce et al. (1994) Int. Ophth. Clinics 34:121-148).
Therefore, there still exists a need for improved reagents that reduce angiogenesis while overcoming the shortcomings of known reagents for inhibiting angiogenesis. Such novel reagents and methods for using them are useful for treating conditions associated with angiogenesis including, without limitation, neoplasia, rheumatoid arthritis, endometriosis, psoriasis, and vascular retinopathies.
The invention provides methods and reagents for inhibiting angiogenesis and allows for the treatment of various angiogenesis-associated conditions.
It has been discovered that cyclin dependent kinase inhibitors (CDKi""s) can inhibit angiogenesis. This discovery has been exploited to develop the present invention which includes methods and compositions for inhibiting angiogenesis.
In a first aspect, the invention provides a method for inhibiting angiogenesis comprising introducing into a target endothelial cell an effective amount of a recombinant virus that comprises a transgene encoding a cyclin dependent kinase inhibitor (CDKi), wherein proliferation and/or migration of the endothelial cell is inhibited. In a certain preferred embodiment, the cyclin dependent kinase inhibitor is derived from a mammal (e.g., a human). In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the CIP/KIP family or an active fragment thereof, such as an active fragment selected from the group consisting of amino acids 25-93 of human p27 protein (SEQ ID NO:26) and amino acids 12-178 of human p27 protein. In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the INK4 family or an active fragment thereof, such as human p1116 protein (SEQ ID NO:28) or an active fragment thereof. In a certain embodiment, the transgene encodes a cyclin dependent kinase inhibitor which can be internalized by a cell. In certain embodiments, the cyclin dependent kinase inhibitor is secretable.
In a certain preferred embodiment, the transgene encodes a cyclin dependent kinase inhibitor which is a fusion protein comprising at least an active fragment of a first cyclin dependent kinase inhibitor and at least an active fragment of a second cyclin dependent kinase inhibitor. In a certain embodiment, the first cyclin dependent kinase inhibitor is a protein from the CIP/KIP family. In certain embodiments, the first cyclin dependent kinase inhibitor is human p27. In a certain embodiment, the fusion protein comprises an active fragment of a protein from the CIP/KIP family, wherein the active fragment is selected from the group consisting of amino acids 25-93 of human p27 protein and amino acids 12-178 of human p27 protein. In a certain embodiment, the second cyclin dependent kinase inhibitor is a protein from the INK4 family. In a certain embodiment, the second cyclin dependent kinase inhibitor is human p1 16. In some embodiments, the cyclin dependent kinase inhibitor is a fusion protein comprising at least an active fragment of human p16 and at least an active fragment of human p27, and is W3 (SEQ ID NO:4), W4 (SEQ ID NO:6), W5 (SEQ ID NO:8), W6 (SEQ ID NO:10), W7 (SEQ ID NO:14), W8 (SEQ ID NO:16), W9 (SEQ ID NO:20), or W10 (SEQ ID NO:22). In preferred embodiments, the cyclin dependent kinase inhibitor is W7 or W9. In a particularly preferred embodiment, the cyclin dependent kinase inhibitor is W9. In a certain embodiment, the fusion protein comprises a linker positioned between the active fragment of the first cyclin dependent kinase inhibitor and the active fragment of the second cyclin dependent kinase inhibitor.
In a preferred embodiment, the recombinant virus is an adenovirus, a lentivirus, a retrovirus, an SV-40 virus, an Epstein Barr virus, a herpesvirus, an adeno-associated virus, or a pox virus, such as a vaccinia virus. In a certain embodiment, the adenovirus lacks an essential viral protein-encoding sequence. In a certain embodiment, the adenovirus is replication-deficient. In one embodiment, the replication-deficient recombinant virus lacks a functional E1 region. In a certain embodiment, the adenovirus lacking the functional E1 region additionally lacks a functional second region, such as an E2 region, an E3 region, or an E4 region.
In a certain embodiment of the first aspect of the invention, the endothelial cell is in a mammal. In a certain embodiment, the mammal is afflicted with a condition associated with angiogenesis, which causes proliferation and/or migration of endothelial cells. In certain embodiments, the condition is neoplasia, rheumatoid arthritis, psoriasis, vascular retinopathy, or endometriosis. In one embodiment, the condition is neoplasia. In the case of neoplasia, the angiogenesis is stimulated by factors produced by a tumor.
In a second aspect, the invention provides a method for inhibiting angiogenesis comprising contacting an endothelial cell with an effective amount of a liposome that comprises a transgene encoding a mammalian cyclin dependent kinase inhibitor, wherein the transgene is internalized by the endothelial cell, wherein proliferation and/or migration of the contacted endothelial cell is inhibited. In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the CIP/KIP family or an active fragment thereof, such as an active fragment selected from the group consisting of amino acids 25-93 of human p27 protein and amino acids 12-178 of human p27 protein. In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the INK4 family or an active fragment thereof, such as human p16 protein or an active fragment thereof.
In a certain embodiment of the second aspect of the invention, the cyclin dependent kinase inhibitor is a fusion protein comprising at least an active fragment of a first cyclin dependent kinase inhibitor and at least an active fragment of a second cyclin dependent kinase inhibitor. In a certain embodiment, the first cyclin dependent kinase inhibitor is a protein from the CIP/KIP family. In certain embodiments, the first cyclin dependent kinase inhibitor is human p27. In a certain embodiment, the fusion protein comprises an active fragment of a protein from the CIP/KIP family, wherein the active fragment is selected from the group consisting of amino acids 25-93 of human p27 protein and amino acids 12-178 of human p27 protein. In a certain embodiment, the second cyclin dependent kinase inhibitor is a protein from the INK4 family. In a certain embodiment, the second cyclin dependent kinase inhibitor is human p16. In some embodiments, the cyclin dependent kinase inhibitor is a fusion protein comprising at least an active fragment of human p16 and at least an active fragment of human p27, and is W7 or W9.
In certain embodiments of the second aspect of the invention, the liposome contains on its external surface a molecule that binds to a cell surface protein on the endothelial cell, wherein binding of the molecule to the cell surface protein facilitates the internalization. In some embodiments, the cyclin dependent kinase inhibitor is internalizable. In certain embodiments, the internalizable cyclin dependent kinase inhibitor is secretable.
In a third aspect, the invention provides a method for inhibiting angiogenesis, comprising contacting a target endothelial cell with a mammalian internalizable cyclin dependent kinase inhibitor, wherein proliferation and/or migration of the target endothelial cell is inhibited, and wherein the inhibitor is selected from the group consisting of a protein from the INK4 family or an active fragment thereof, a protein from the CIP/KIP family or an active fragment thereof, and a fusion protein comprising at least an active fragment of the protein from the INK4 family and at least an active fragment of the protein from the CIP/KIP family. In some embodiments, the active fragment of the protein from the CIP/KIP family is amino acids 25-93 of human p27 protein or amino acids 12-178 of human p27 protein. In one embodiment, the cyclin dependent kinase inhibitor is W7 or W9. In a preferred embodiment, the cell internalizes the cyclin dependent kinase inhibitor. In certain embodiments, the method further comprises delivering a transgene encoding the cyclin dependent kinase inhibitor to an auxiliary cell, wherein the transgene is expressed by the auxiliary cell to produce the cyclin dependent kinase inhibitor, wherein the auxiliary cell releases the cyclin dependent kinase inhibitor into the blood and wherein the bloodborne cyclin dependent kinase inhibitor contacts the target endothelial cell. In a certain embodiment, the cyclin dependent kinase inhibitor comprises a secretable segment and the auxiliary cell releases the cyclin dependent kinase inhibitor by secretion.
In a fourth aspect, the invention provides a purified internalizable form of a cyclin dependent kinase inhibitor. In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the CIP/KIP family or an active fragment thereof, such as an active fragment selected from the group consisting of amino acids 25-93 of human p27 protein and amino acids 12-178 of human p27 protein. In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the INK4 family or an active fragment thereof, such as human p16 protein or an active fragment thereof. In a certain preferred embodiment, the cyclin dependent kinase inhibitor is derived from a mammal (e.g., a human).
In a certain embodiment of the fourth aspect of the invention, the purified internalizable form of the cyclin dependent kinase inhibitor is a fusion protein comprising at least an active fragment of a first cyclin dependent kinase inhibitor and at least an active fragment of a second cyclin dependent kinase inhibitor. In a certain embodiment, the first cyclin dependent kinase inhibitor is a protein from the CIP/KIP family. In certain embodiments, the first cyclin dependent kinase inhibitor is human p27. In a certain embodiment, the fusion protein comprises an active fragment of a protein from the CIP/KIP family, wherein the active fragment is selected from the group consisting of amino acids 25-93 of human p27 protein and amino acids 12-178 of human p27 protein. In a certain embodiment, the second cyclin dependent kinase inhibitor is a protein from the INK4 family. In a certain embodiment, the second cyclin dependent kinase inhibitor is human p16. In some embodiments, the cyclin dependent kinase inhibitor is a fusion protein comprising at least an active fragment of human p16 and at least an active fragment of human p27, and is W7 or W9. In a particularly preferred embodiment, the cyclin dependent kinase inhibitor is W9. In a certain embodiment, the fusion protein comprises a linker positioned between the active fragment of the first cyclin dependent kinase inhibitor and the active fragment of the second cyclin dependent kinase inhibitor.
In a fifth aspect, the invention provides a method for treating a condition associated with angiogenesis. In this method, a therapeutically effective amount of a therapeutic composition comprising a purified internalizable form of a cyclin dependent kinase inhibitor and a pharmaceutically acceptable carrier is administered to a patient having or suspected of having the condition.
In a sixth aspect, the invention provides a method for treating a condition associated with angiogenesis wherein a therapeutically effective amount of a recombinant virus comprising a transgene encoding a cyclin dependent kinase inhibitor is administered to a patient having or suspected of having the condition. In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the CIP/KIP family or an active fragment thereof, such as an active fragment selected from the group consisting of amino acids 25-93 of human p27 protein and amino acids 12-178 of human p27 protein. In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the INK4 family or an active fragment thereof, such as human p16 protein or an active fragment thereof. In a certain preferred embodiment, the cyclin dependent kinase inhibitor is a derived from a mammal (e.g., a human).
In a certain embodiment of the sixth aspect of the invention, the cyclin dependent kinase inhibitor is a fusion protein comprising at least an active fragment of a first cyclin dependent kinase inhibitor and at least an active fragment of a second cyclin dependent kinase inhibitor. In a certain embodiment, the first cyclin dependent kinase inhibitor is a protein from the CIP/KIP family. In certain embodiments, the first cyclin dependent kinase inhibitor is human p27. In a certain embodiment, the fusion protein comprises an active fragment of a protein from the CIP/KIP family, wherein the fragment is selected from the group consisting of amino acids 25-93 of human p27 protein and amino acids 12-178 of human p27 protein. In a certain embodiment, the second cyclin dependent kinase inhibitor is a protein from the INK4 family. In a certain embodiment, the second cyclin dependent kinase inhibitor is human p16. In some embodiments, the cyclin dependent kinase inhibitor is a fusion protein comprising at least an active fragment of human p16 and at least an active fragment of human p27, and is W7 or W9. In a particularly preferred embodiment, the cyclin dependent kinase inhibitor is W9. In a certain embodiment, the fusion protein comprises a linker positioned between the active fragment of the first cyclin dependent kinase inhibitor and the active fragment of the second cyclin dependent kinase inhibitor.
In a particular embodiment of the sixth aspect of the invention, the recombinant virus is an adenovirus, a lentivirus, a retrovirus, an SV-40 virus, an Epstein Barr virus, a herpesvirus, an adeno-associated virus, or a pox virus, such as a vaccinia virus. In a certain embodiment, the adenovirus lacks an essential viral protein-encoding sequence. In a certain embodiment, the adenovirus is replication-deficient, preferably because it lacks a functional E1 region. In a certain embodiment, the adenovirus lacking the functional E1 region additionally lacks a functional second region, such as an E2 region, an E3 region, or an E4 region. In a certain embodiment, the recombinant virus expresses on its external surface a molecule that binds to a cell surface protein on the endothelial cell, wherein binding of the molecule to the cell surface protein facilitates the transduction of the endothelial cell by the recombinant virus.
In a seventh aspect, the invention provides a recombinant virus comprising a transgene encoding a cyclin dependent kinase inhibitor, wherein the recombinant virus is an adenovirus lacking an E1 region, a lentivirus, a retrovirus, an SV-40 virus, an Epstein Barr virus, a herpesvirus, an adeno-associated virus, or a pox virus, such as a vaccinia virus. In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the CIP/KIP family or an active fragment thereof, such as an active fragment selected from the group consisting of amino acids 25-93 of human p27 protein and amino acids 12-178 of human p27 protein. In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the INK4 family or an active fragment thereof, such as human p16 protein or an active fragment thereof. In a certain preferred embodiment, the cyclin dependent kinase inhibitor is a derived from a mammal (e.g., a human).
In a certain embodiment of the seventh aspect of the invention, the cyclin dependent kinase inhibitor is a fusion protein comprising at least an active fragment of a first cyclin dependent kinase inhibitor and at least an active fragment of a second cyclin dependent kinase inhibitor. In a certain embodiment, the first cyclin dependent kinase inhibitor is a protein from the CIP/KIP family. In certain embodiments, the first cyclin dependent kinase inhibitor is human p27. In a certain embodiment, the fusion protein comprises an active fragment of a protein from the CIP/KIP family, wherein the fragment is selected from the group consisting of amino acids 25-93 of human p27 protein and amino acids 12-178 of human p27 protein. In a certain embodiment, the second cyclin dependent kinase inhibitor is a protein from the INK4 family. In a certain embodiment, the second cyclin dependent kinase inhibitor is human p16. In some embodiments, the cyclin dependent kinase inhibitor is a fusion protein comprising at least an active fragment of human p16 and at least an active fragment of human p27, and is W7 or W9. In a particularly preferred embodiment, the cyclin dependent kinase inhibitor is W9. In a certain embodiment, the fusion protein comprises a linker positioned between the active fragment of the first cyclin dependent kinase inhibitor and the active fragment of the second cyclin dependent kinase inhibitor.
In an eighth aspect, the invention provides a liposome comprising a cyclin dependent kinase inhibitor. In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the CIP/KIP family or an active fragment thereof, such as an active fragment selected from the group consisting of amino acids 25-93 of human p27 protein and amino acids 12-178 of human p27 protein. In a certain embodiment, the cyclin dependent kinase inhibitor is a protein from the INK4 family or an active fragment thereof, such as human p16 protein or an active fragment thereof. In a certain preferred embodiment, the cyclin dependent kinase inhibitor is a derived from a mammal (e.g., a human).
In a certain embodiment of the eighth aspect of the invention, the cyclin dependent kinase inhibitor is a fusion protein comprising at least an active fragment of a first cyclin dependent kinase inhibitor and at least an active fragment of a second cyclin dependent kinase inhibitor. In a certain embodiment, the first cyclin dependent kinase inhibitor is a protein from the CIP/KIP family. In certain embodiments, the first cyclin dependent kinase inhibitor is human p27. In a certain embodiment, the fusion protein comprises an active fragment of a protein from the CIP/KIP family, wherein the fragment is selected from the group consisting of amino acids 25-93 of human p27 protein and amino acids 12-178 of human p27 protein. In a certain embodiment, the second cyclin dependent kinase inhibitor is a protein from the INK4 family. In a certain embodiment, the second cyclin dependent kinase inhibitor is human p16. In some embodiments, the cyclin dependent kinase inhibitor is a fusion protein comprising at least an active fragment of human p16 and at least an active fragment of human p27, and is W7 or W9. In a particularly preferred embodiment, the cyclin dependent kinase inhibitor is W9. In a certain embodiment, the fusion protein comprises a linker positioned between the active fragment of the first cyclin dependent kinase inhibitor and the active fragment of the second cyclin dependent kinase inhibitor.
In a ninth aspect, the invention provides a nucleic acid composition comprising a nucleic acid sequence encoding an internalizable form of a cyclin dependent kinase inhibitor, wherein the nucleic acid sequence is operably linked to a regulatory sequence which regulates cellular expression of the nucleic acid sequence. In one embodiment, the nucleic acid sequence encodes a secretion signal.
In a tenth aspect, the invention provides a therapeutic composition comprising a nucleic acid sequence encoding an internalizable form of a cyclin dependent kinase inhibitor, wherein the nucleic acid sequence is operably linked to a regulatory sequence which regulates cellular expression of the nucleic acid sequence, and a pharmaceutically acceptable carrier. In one embodiment, the therapeutic composition further comprises a delivery system that facilitates the internalization of the composition by an endothelial cell. In certain embodiments, the delivery system is a liposome comprising the nucleic acid composition. In certain embodiments, the delivery system is a recombinant virus comprising the nucleic acid composition. In preferred embodiments, the recombinant virus is an adenovirus, a retrovirus, an SV-40 virus, an Epstein Barr virus, a herpesvirus, an adeno-associated virus, or a pox virus. In one embodiment, the nucleic acid sequence also encodes a secretion signal.
In an eleventh aspect, the invention provides a method for treating a patient having or suspected of having a condition associated with angiogenesis comprising delivering a therapeutically effective amount of a transgene encoding a secretable, internalizable form of a cyclin dependent kinase inhibitor to cells of the patient that are in close proximity to endothelial cells affected by the condition. In one embodiment, the therapeutic composition further comprises a delivery system that facilitates the internalization of the composition by an endothelial cell. In certain embodiments, the delivery system is a liposome comprising the transgene. In certain embodiments, the delivery system is a recombinant virus comprising the transgene. In preferred embodiments, the recombinant virus is an adenovirus, a retrovirus, an SV-40 virus, an Epstein Barr virus, a herpesvirus, an adeno-associated virus, or a pox virus.